The major focus of the research is the study of reaction mechanisms of the oxidizing enzymes at a fundamental level by using laser flash photolysis (LFP) methods to produce reactive oxidants in models and in the enzymes and related studies with mechanistic probe substrates that provide details about the transients formed in the reactions. The primary targets of study are the P450 enzymes, which have numerous disease relationships and are among the more important enzymes for the pharmaceutical industry. Two new LFP methods, photo-induced ligand cleavage of iron(IV) complexes and photo-oxidation of iron(IV)-oxo species, are employed to produce various high-valent iron-oxo transients that are the putative reactive species in the enzymes. This methodology permits kinetic studies on the microsecond time scale, which is 4-5 orders of magnitude faster than can be achieved in rapid mixing experiments. Specific aims include production of the elusive iron-oxo species in P450 enzymes via photo-oxidation of Compound II species, formation and characterization of high valent iron-oxo species in models and, if possible, in heme- containing enzymes, kinetic characterization of various iron-oxo species in models and in enzymes to evaluate the kinetic competency in P450-catalyzed oxidation reactions, extensions of the methods to di-iron containing enzymes as a long-range goal of the program, and mechanistic probe studies designed to test for radical and/or cationic intermediates formed in the enzyme-catalyzed reactions. The P450 enzymes are among the more important enzymes involved in cancer and liver disease and are associated with numerous other diseases, and control of P450 enzymes is an important therapeutic goal and a concern of the pharmaceutical industry. A better understanding of the fundamental nature of the reactive species formed in the enzymes and the mechanisms of their reactions is expected to lead to lead ultimately to health-related advances in disease treatment and drug design.